Byl719 (alpelisib) for use in the treatment of pik3ca-related overgrowth spectrum (pros-cloves syndrome)

ABSTRACT

The present invention relates to a method of treating PIK3CA-Related Overgrowth Spectrum (PROS) more particularly, Congenital, Lipomatous, Overgrowth, Vascular Malformations, Epidermal Nevi and Spinal/Skeletal Anomalies and/or Scoliosis (CLOVES) 10 syndrome. To date, there are no specific treatments for patients and no animal models of PROS to better understand the physiopathology of the disorder. Inventors developed a genetic mouse model of PROS that recapitulates the human disease and demonstrated the efficacy of BYL719. Based on these results they treated two patients, one adult and one child, with severe CLOVES syndrome using BYL719. The drug had a robust efficiency on disease in the 15 two patients inducing quick recovery of all affected organs. Thus, the invention relates to a method of treating PROS in a subject in need thereof comprising the step of administrating the subject with a therapeutically effective amount of BYL719.

FIELD OF THE INVENTION

The invention relates to methods for the treatment of PIK3CA-relatedovergrowth spectrum (PROS), such as congenital lipomatous overgrowth,vascular malformations and epidermal nevi (CLOVES).

BACKGROUND OF THE INVENTION

The term “PROS” for PIK3CA-Related Overgrowth Spectrum” was agreed uponto encompass both the known and emerging clinical entities associatedwith somatic PIK3CA mutations including, macrodactyly, FAO, HHML,CLOVES, and related megalencephaly conditions (Keppler-Noreuil et al2014). A workshop was convened at the National Institutes of Health(NIH) to discuss and develop a consensus document regarding diagnosisand treatment of patients with PIK3CA-associated somatic overgrowthdisorders.

CLOVES, stands for Congenital, Lipomatous, Overgrowth, VascularMalformations, Epidermal Nevi and Spinal/Skeletal Anomalies and/orScoliosis. This syndrome is considered as a rare disorder andcharacterised by progressive, complex, and mixed truncal vascularmalformations, dysregulated adipose tissue, varying degrees ofscoliosis, and enlarged bony structures without progressive bonyovergrowth (Sapp et al 2007; Alomari et al 2009). This syndrome isdifferent from cancer. In fact, in CLOVES syndrome, tumours are benign,the tissues are just overgrowth and the subject is deformed. CLOVESsyndrome is rare and evident at birth. It affects males and femalesequally regardless of their race or ethnicity. Many of the patients withthis syndrome are misdiagnosed.

CLOVES syndrome is caused by a somatic mosaic mutation in PIK3CA gene.PIK3CA encodes the 110-kD catalytic alpha subunit of PI3K, which inresponse to tyrosine kinase receptor ligand binding is activated andconverts phosphatidylinositol (3,4)-bisphosphate (PIP2) tophosphatidylinositol (3,4,5)-triphosphate (PIP3). Activating mutationsin PIK3CA have been described in several types of cancers but never inCLOVES syndrome. In 2012, Kurek et al has identified the activatingmutations in PIK3CA by sequencing DNA or RNA. But, today, there is nocure for CLOVES Syndrome.

Limaye et al 2015 discloses that somatic mutations in PIK3CA areinvolved in cancers, overgrowth syndromes, and lymphatic malformation(LM). More particularly, the authors showed that cultured cell linesderived from healthy donors (not affected by PROS), called humanumbilical venous endothelial cells (HUVEC) then retrovirally transfectedwith mutant PIK3CA have an activation of the AKT/mTORC pathway. The drugBYL719 abolished, as expected, PIK3CA-variant induced AKTphosphorylation in this artificial model, indicating that these proteinsparticipate in the same signalling pathway. But, this in vitro modeldoes not recapitulate any symptom or disease phenotype related to PROSpatients, more particularly does not show any evidence that this drugcould be used to treat patients suffering from PROS. Thus, there is aneed to understand the use of BYL719 in patients with PROS, moreparticularly with CLOVES syndrome or Klippel-Trenaunay syndrome.

SUMMARY OF THE INVENTION

The present invention relates to a method of treating PROS in a subjectin need thereof comprising a step of administrating the subject with atherapeutically effective amount of BYL719. In particular, the presentinvention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

BYL719 synthesized by Novartis is in clinical trial at phase II/III foradvanced solid tumours. Inventors developed the first genetic mousemodel of PROS that recapitulates the human disease and demonstrated theefficacy of BYL719, a pharmacological inhibitor of PIK3CA, in preventingand improving all organs dysfunction in the PROS mouse. Based on theseresults they treated two patients, one adult and one child, with severeCLOVES syndrome using BYL719. The drug had a robust efficiency ondisease in the two patients inducing quick recovery of all affectedorgans. Previously intractable vascular tumors shrunk, congestive heartfailure fully recovered, hemi hypertrophy reduced, and scoliosis wasattenuated. The drug was not associated with any significant sideeffects. In conclusion, this study provides the first direct evidencesupporting PIK3CA inhibition as a promising therapeutic in PROSpatients.

Accordingly, the present invention relates to a method of treating PROSin a subject in need thereof comprising a step of administrating thesubject with a therapeutically effective amount of BYL719. Moreparticularly, the invention relates to a method of treating CLOVESsyndrome in a subject in need thereof comprising the step ofadministrating the subject with a therapeutically effective amount ofBYL719.

As used herein, the terms “treating” or “treatment” refer to bothprophylactic or preventive treatment as well as curative or diseasemodifying treatment, including treatment of subject at risk ofcontracting the disease or suspected to have contracted the disease aswell as subject who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment. By “therapeutic regimen” is meant the pattern oftreatment of an illness, e.g., the pattern of dosing used duringtherapy. A therapeutic regimen may include an induction regimen and amaintenance regimen. The phrase “induction regimen” or “inductionperiod” refers to a therapeutic regimen (or the portion of a therapeuticregimen) that is used for the initial treatment of a disease. Thegeneral goal of an induction regimen is to provide a high level of drugto a subject during the initial period of a treatment regimen. Aninduction regimen may employ (in part or in whole) a “loading regimen”,which may include administering a greater dose of the drug than aphysician would employ during a maintenance regimen, administering adrug more frequently than a physician would administer the drug during amaintenance regimen, or both. The phrase “maintenance regimen” or“maintenance period” refers to a therapeutic regimen (or the portion ofa therapeutic regimen) that is used for the maintenance of a subjectduring treatment of an illness, e.g., to keep the subject in remissionfor long periods of time (months or years). A maintenance regimen mayemploy continuous therapy (e.g., administering a drug at a regularintervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy(e.g., interrupted treatment, intermittent treatment, treatment atrelapse, or treatment upon achievement of a particular predeterminedcriteria [e.g., pain, disease manifestation, etc.]).

As used herein the term “PROS” refers to PIK3CA-Related OvergrowthSpectrum. It is a group of disorders such as fibroadipose overgrowth(FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenitallipomatous asymmetric overgrowth of the trunk, lymphatic, capillary,venous, and combined-type vascular malformations, epidermal nevi,skeletal and spinal anomalies (CLOVES) syndrome and HemihyperplasiaMultiple Lipomatosis (HHML) and Klippel-Trenaunay syndrome.

As used herein, the term “fibroadipose overgrowth (FAO)” refers to asyndrome, which is characterized by the major findings of segmentalprogressive overgrowth of subcutaneous, muscular, and visceralfibroadipose tissue with skeletal overgrowth (Lindhurst et al 2012).

As used herein, the term “megalencephaly-capillary malformation (MCAP)syndrome” refers to a syndrome which is characterized by the majorfindings of (1) megalencephaly (MEG) or hemimegalencephaly (HMEG)associated with neurologic findings of hypotonia, seizures, and mild tosevere intellectual disability; and (2) cutaneous capillarymalformations with focal or generalized somatic overgrowth (Mirzaa et al2013).

In a particular embodiment, the PROS disorder is CLOVES. As used herein,the term “CLOVES” refers to Congenital, Lipomatous, Overgrowth, VascularMalformations, Epidermal Nevi and Spinal/Skeletal Anomalies and/orScoliosis. This syndrome is characterised by lipomatous tissues showingcomplex congenital overgrowth (typically appearing as a truncallipomatous mass) and a combination of vascular and lymphaticmalformations.

As used herein, the term “Hemihyperplasia Multiple Lipomatosis (HHML)”refers to a condition characterized by asymmetric nonprogressiveovergrowth, multiple lipomas, and superficial vascular malformations (BG et al 2013).

In a particular embodiment, the PROS disorder is Klippel-Trenaunaysyndrome. As used herein, the term “Klippel-Trenaunay syndrome” refersto a rare congenital medical condition in which blood vessels and/orlymph vessels fail to form properly.

Thus, the method according to the present invention can be supplied to asubject, who has been diagnosed as presenting one of the disorders inPROS.

As used herein, the term “subject” refers to any mammals, such as arodent, a feline, a canine, and a primate. Particularly, in the presentinvention, the subject is a human afflicted with or susceptible to beafflicted with PROS disorders. In a particular embodiment, the subjectis a human afflicted or susceptible to be afflicted with CLOVESsyndrome. In a particular embodiment, the subject is a human afflictedor susceptible to be afflicted with Klippel-Trénaunay syndrome.

As used herein, the term “BYL719” is an ATP-competitive oral PI3Kinhibitor selective for the p110α isoform that is activated by a mutantPIK3CA gene (Furet P., et al. 2013; Fritsch C., et al 2014). Thismolecule is also called Alpelisib and has the following formula in theart:

A “therapeutically effective amount” is intended for a minimal amount ofactive agent which is necessary to impart therapeutic benefit to asubject. For example, a “therapeutically effective amount” to a subjectis such an amount which induces, ameliorates or otherwise causes animprovement in the pathological symptoms, disease progression orphysiological conditions associated with or resistance to succumbing toa disorder. It will be understood that the total daily usage of thecompounds of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular subject willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; activity of the specific compoundemployed; the specific composition employed, the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidential with the specific compound employed; and like factors wellknown in the medical arts. For example, it is well within the skill ofthe art to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. However, thedaily dosage of the products may be varied over a wide range from 0.01to 1,000 mg per adult per day. Typically, the compositions contain 0.01,0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500mg of the active ingredient for the symptomatic adjustment of the dosageto the subject to be treated. A medicament typically contains from about0.01 mg to about 500 mg of the active ingredient, preferably from 1 mgto about 100 mg of the active ingredient. An effective amount of thedrug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7mg/kg of body weight per day.

The PIK3CA inhibitor as described above may be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form pharmaceuticalcompositions. “Pharmaceutically” or “pharmaceutically acceptable” referto molecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to a mammal,especially a human, as appropriate. A pharmaceutically acceptablecarrier or excipient refers to a non-toxic solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. The pharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, transdermal, localor rectal administration, the active principle, alone or in combinationwith another active principle, can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports, to animals and human beings. Suitable unit administrationforms comprise oral-route forms such as tablets, gel capsules, powders,granules and oral suspensions or solutions, sublingual and buccaladministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and intranasal administration forms and rectaladministration forms. Typically, the pharmaceutical compositions containvehicles which are pharmaceutically acceptable for a formulation capableof being injected. These may be in particular isotonic, sterile, salinesolutions (monosodium or disodium phosphate, sodium, potassium, calciumor magnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions. The pharmaceutical forms suitablefor injectable use include sterile aqueous solutions or dispersions;formulations including sesame oil, peanut oil or aqueous propyleneglycol; and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases, the form mustbe sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Solutions comprisingcompounds of the invention as free base or pharmacologically acceptablesalts can be prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms. The polypeptide(or nucleic acid encoding thereof) can be formulated into a compositionin a neutral or salt form. Pharmaceutically acceptable salts include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. The carrier can also be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetables oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminium monostearate and gelatin. Sterileinjectable solutions are prepared by incorporating the activepolypeptides in the required amount in the appropriate solvent withseveral of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. Upon formulation, solutions will be administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed. For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved in 1 mlof isotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: Characterization of the mouse model of PROS and efficacy of theBYL719. A) Kaplan-Meier survival curves of PIK3CA^(WT) andPIK3CA^(CAGG-CreER) mice after tamoxifen administration. B) Kaplan-Meiersurvival curves of PIK3CA^(CAGG-CreER) mice treated or not with BYL719after tamoxifen administration. After 40 days of treatment the BYL719 iswithdrawn. C) Kaplan-Meier survival curves of PIK3CA^(CAGG-CreER) micetreated or not with BYL719 10 days after tamoxifen administration. Scalebars 10 μm.

FIG. 2: Efficiency of the BYL719 in an adult patient with severe CLOVESsyndrome. A) Clinical parameters (weight, chest girth and waistcircumferences) of the patient 1 before and after BYL719 initiation. B)Brain Natriuretic Peptide (BNP) and serum creatinine levels before andafter BYL719 initiation.

FIG. 3: Efficiency of the BYL719 in a child patient with severe CLOVESsyndrome. Clinical parameters (thigh and waist circumferences) of thepatient 2 before and after BYL719 initiation.

EXAMPLE

Material & Methods Animals

For this study, we interbedded homozygous R26Stop^(FL)P110* (Stock#012343) and heterozygous CAGGCre-ER^(TM) (Stock #004682) on C57BL/6background obtained from The Jackson Laboratories. We obtainedR26Stop^(FL)P110*^(−/−)×CAGGCreER^(TM+) (referred here asPIK3CA^(CAGG-CreER)) and R26Stop^(FL)P110*^(+/−) ×CAGGCre-ER^(TM−)(referred here as PIK3CA^(WT)). Animals were fed ad libitum and housedat constant ambient temperature in a 12-hour light cycle. Animalprocedures were approved by the Departmental Director of “ServicesVétérinaires de la Préfecture de Police de Paris” and by the ethicalcommittee of the Paris Descartes University. A single dose of tamoxifen(40 mg×kg⁻¹) was administered through oral gavage at the age of 21 days.For survival studies, mice were followed daily after tamoxifen gavage(PIK3CA^(WT) n≈16) n=16 and PIK3CA^(CAGG-CreER) For therapeutic studies,mice were treated with the PI3KCA inhibitor BYL719 (Chem Express; 50mg×kg⁻¹ in 0.5% carboxymethylcellulose (Sigma), daily p.o.) or vehicle(0.5% carboxymethylcellulose (Sigma), daily p.o.). Treatment was startedin the meantime of tamoxifen gavage for the preventive study(PIK3CA^(CAGG-CreER) n=18) or ten days after for the therapeutic study(PIK3CA^(CAGG-CreER) n=6). A total of 6 mice were sacrificed on day 51after tamoxifen gavage in the vehicle group of the PIK3CA^(CAGG-CreER),6 mice on day 51 after tamoxifen gavage in the preventive BYL719 groupand 6 mice on day 70 after tamoxifen gavage in the therapeutic study ofthe PIK3CA^(CAGG-CreER) for tissues examination.

Cell Cultures

Breast cancer cell lines T-47D were obtained from Sigma Aldrich. Cellswere cultured in media with DMEM+2 mM Glutamine+10% Fetal Bovine Serum(FBS). For BYL719 experiments (Chem Express), cells were treated withincreasing concentration of BYL719 (0, 0.5, 1 and 5 μmol/L) for 2, 4 and6 hours before western blot was performed. Each experiment was performedin duplicate and repeated at least three times.

Morphological Analysis

Mousse tissues were fixed in 4% paraformaldehyde, paraffin embedded.4-μm sections of liver were stained with periodic acid Schiff (PAS),4-μm sections of liver of spleen were stained with hematoxylin and eosin(H&E) and 4-μm sections of kidneys were stained with Masson's trichrome.

Immunohistochemistry and Immunofluorescence

4-μm sections of paraffin-embedded kidneys were incubated withanti-P-AKT (Ser⁴⁷³) antibody (Cell Signaling Technology, ref #4060),anti-P-S6RP antibody (Cell Signaling Technology, ref #5364) andanti-CD34 antibody (eBioscience, ref #14-0341). Immunofluorescencestudies were analyzed using the confocal microscope Zeiss LSM 700.

Western Blot

Western blots were performed as previously described²¹. Briefly, proteinextracts from liver, muscles, heart, kidneys and T-47D cells wereresolved by SDS-PAGE before being transferred onto the appropriatemembrane and incubated with anti-P-AKT (Ser⁴⁷³) antibody (Cell SignalingTechnology, ref #4060), anti-P-AKT (Thr³⁰⁸) antibody (Cell SignalingTechnology, ref #13038), anti-P-S6RP antibody (Cell SignalingTechnology, ref #5364), anti-GAPDH (Merck Millipore, ref #374) andanti-β actin antibody (Sigma-Aldrich, ref # A2228), followed by theappropriate peroxidase-conjugated secondary antibody. Chemiluminescencewas acquired using a Fusion FX7 camera (Vilbert Lourmat) anddensitometry was performed using BiolD software (Certain Tech).

Patients

This study was performed in the Renal Division of Necker Hospital,starting in September 2015. The patient with the CLOVES syndrome hadclinical examination, tumor measurements, naevi measurement beforetreatement instauration. BYL719 treatment was initiated at the dose of250 mg orally per day. The study was conducted on two patients, an adultand a child, followed at Necker hospital. This protocol was approved bythe ANSM (authorization n° 553984-986 and n° 584018); informed writtenconsent was obtained from each patient or their legal representative.BYL719 was compassionately offered by Novartis. The first patientreceived 250 mg/day and the second patient 50 mg/day. BYL719 was orallydelivered every morning before breakfast. Glycaemia was monitored afterany meal during two months and then progressively sparse.

Data Analysis and Statistics

Data were expressed as means±SEM. Survival curves were analyzed with aMantel-Cox (log-rank) test. Differences between the experimental groupswere evaluated using ANOVA, followed when significant (P<0.05) by theTukey-Kramer test. When only two groups were compared, Mann-Whitneytests were used. The statistical analysis was performed using GraphPrism Software.

Renal Function

The serum creatinine level was measured weekly during the first year andevery 3 months thereafter using a Synchron Cx4 autoanalyzer (BeckmanCoulter, Villepinte, France). The glomerular filtration rate wasestimated using the MDRD formula (eGFR).

Biopsy Samples and Morphological Analysis

Kidney biopsy specimens were fixed in alcohol-formalin-acetic acidsolution and embedded in paraffin. Four-micrometer sections were stainedwith the Periodic Acid Schiff (PAS) stain, Masson's trichrome andhematoxylin and eosin (H&E). Electron microscopy analyses wereperformed.

Immunohistochemistry and Immunofluorescence

Four sections of paraffin-embedded kidneys were incubated withanti-nephrin antibody (Progen), anti-WT1 antibody (Dako), anti-podocinantibody (Sigma) and anti-synaptopodin antibody (Novus Biologicals).Upon the total glomerular area. For each biopsies, all glomerularsection were quantified. The primary antibodies were revealed with theappropriate Alexa 488- or 555-conjugated secondary antibodies (MolecularProbes). Immunofluorescence staining was visualized using the Zeiss LSM700 confocal microscope. The podocyte-stained area was automaticallyquantified using a Nikon digital camera Dx/m/1200 and Image J softwareand expressed as the percentage of the podocyte-stained area.

In Situ Hybridization

Alcohol-formalin-acetic acid solution-fixed, paraffin-embedded tissueswere assayed for PI3KCα RNA expression using a previously describeddigoxygenin-anti-digoxygenin technique. Nitrobluetetrazolium-5-bromo-4-chloro-3-indolyl phosphate toluidinium (NBT-BCIP)was used to visualize infected cells in the tissues. The specificity ofthe hybridization signal was systematically checked by hybridizing senseprobes with parallel sections and anti-sense probes with uninfectedrenal tissues. ISH-stained tissues were visualized and photographed withan Olympus Proxis microscope and a Zeiss Axio Cam ICcl.

Cell Culture

Human fibroblasts were grown in DMEM supplemented with 15% FBS,penicillin (50 IU/ml)/streptomycin (50 μg/ml), and nonessential aminoacids (Invitrogen).

Western Blot

Western blot were performed as previously described, using a rabbitantibody to human phospho-p70 S6 kinase (Thr389) (Cell SignalingTechnologies) at 1:1000 followed by a horseradish peroxidase-conjugatedantirabbit secondary antibody at 1:10,000 (Dako). The phosphorylationstatus of the p70 S6 kinase at the Thr389 site is specific for mTORphosphorylation. A mouse monoclonal anti-b-actin antibody(Sigma-Aldrich, Lyon, France) was used as control. Proteinphosphorylation levels were normalized to the matching densitometricvalues of b-actin.

DNA Sequencing

DNA was extracted using standard techniques from peripheral bloodmononuclear cells (PBMCs) taken from the patient and skin biopsy in thetumor area. Mutation screening was performed by direct sequencing of allthe PI3KCα exons and flanking intronic regions.

Volumetric Determination

Magnetic resonance imaging (MRI) was used to determine the volume ofeach tumor. MRI was performed at day 0 before BYL719 initiation and thenmonthly. In brief, the volume was calculated by summing the products ofthe area measurements and slice thickness.

Data Analysis and Statistics

The data were expressed as the means±SD. Differences among theexperimental groups were evaluated using ANOVA, followed whensignificant by the Tukey-Kramer test. When only two groups werecompared, the Mann-Whitney test was used. Probability values <0.05 wereconsidered statistically significant. Analyses were performed withGraphPad Prism 5 (GraphPad software, La Jolla, Calif.).

Results

Inventors have observed a significant reduction of the size of theovergrown tissues in the subject, which was administered with BYL719.After a month of treatment, the patient weight decreased from 83.5 kg to73.5 kg. This weigh loss was related to a dramatic reduction of theoedema and an improvement of the global cardiac function rate (cardiacoutput was 22 l/min in December 2015 before treatment and measured at8l/min on February the 8th). The plasmatic brain natriuretic peptidelevels decreased from 2500 pg/ml to 240 pg/ml at day 30 confirming theimprovement of the heart failure. The subcutaneous tumour sizes showed aglobal reduction of 10 percent after 30 days of treatment as assed bythe CT scan and Magnetic Resonance Imaging (MRI). The Karnofskyperformance status scale of the patient improved from 40 to 60% in 30days. Haemoglobin level increased from 8 g/dl to 11.8 g/dl.

Some skin areas (left ear) showed accelerated age phenotype beforetreatment that were improved after a month of BYL719 administration.Furthermore, several large naevi showed decolouration after thebeginning of the treatment.

A Mouse Model of PROS

We began this study by developing a mouse model of PROS. To this end, wetook advantage of the transgenic mouse strain R26StopFLP110*. These miceallow for inducible expression of activated PI3KCA heterodimer in atissue-specific manner. R26StopFLP110* mice were crossed with CAGG-CreERmice to generate PIK3CA^(CAGG-CreER) animals that ubiquitouslyoverexpress PIK3CA upon tamoxifen administration. Three weeks old micewere treated with a single dose of 40 mg·kg⁻¹ tamoxifen to induce Crerecombination¹⁶. We observed that compared to the control PIK3CA^(WT)mice, the PIK3CA^(CAGG-CreER) mice started to die on day 3 post Creinduction with an average of 6 days post Cre recombination (FIG. 1A).Death occurring suddenly in most cases, with necropsy revealingintraabdominal and hepatic hemorrhages (data not shown). Some micedisplayed difficulty walking with hypertrophic psoas muscle assessed bythe magnetic resonance imaging (MRI) (data not shown). Additionally,whole body MRI showed the rapid occurrence of scoliosis, vesselsabnormalities, kidney cysts, and muscles hypertrophy (FIG. 1C).Histological examination revealed multiple organs abnormalitiesincluding severe liver steatosis with vessel disorganization (data notshown), loss of integrity of the spleen microarchitecture (data notshown), spontaneous hemorrhage, and fibrosis of the kidney with aberrantvessels. To further characterize the vessels abnormalities, we performedCD34 immunostaining that confirmed the presence of severe vesseldilation. As expected, western blot and immunofluorescence studiesshowed AKT/mTORC pathway activation in all examined organs. We thereforeconcluded that the PIK3CA^(CAGG-CreER) mice recapitulate human PROSphenotype.

BYL719 Prevents and is an Effective Therapy for PROS in Mice

We decided to test the impact of BYL719 in PIK3CA^(CAGG-CreER) mice. Tothis aim, we first performed a preventive study where the BYL719 wasorally administrated starting just after Cre induction (data not shown).We observed that daily administration of BYL719 dramatically improvedanimal survival (FIG. 1C). In fact, while all PIK3CA^(CAGG-CreER) micein the placebo group died within 21 days, PIK3CA^(CAGG-CreER) micetreated with BYL719 were alive 40 days later with overtly normalappearance. Importantly, treatment interruption 40 days after Crerecombination lead to the death of all the PIK3CA^(CAGG-CreER) animals.We sacrificed several PIK3CA^(CAGG-CreER) mice receiving BYL719 on day40 after Cre induction. Histological examination showed that micetreated with BYL719 had preserved tissues (data not shown) and normalvessels (data not shown). Western blot and immunofluorescence confirmedthe efficiency of BYL719 at inhibiting PI3KCA activation (data notshown).

Next, we performed a therapeutic study by giving either the placebo orthe BYL719 to PIK3CA^(CAGG-CreER) mice ten days after Cre induction,when mice begin to die (FIG. 1C). On day 10 after Cre induction, MRIconfirmed the presence of tissue abnormalities as described previously(data not shown). While the placebo group mice died within the nextfollowing days, BYL719 dramatically improved the survival ofPIK3CA^(CAGG-CreER) mice (FIG. 1J). MRI performed 12 days after thebeginning of the treatment (22 days after Cre induction) demonstratedvery rapid improvement of scoliosis, muscle hypertrophy, and vesselabnormalities (data not shown). Histological analysis revealed thatBYL719 treated mice displayed no, or only minor, tissue changes (datanot shown). As in the preventive study, western blot andimmunofluorescence confirmed PIK3CA inhibition (data not shown).

We concluded that the BYL719 was able to dramatically improve PROS mice,suggesting it may be an effective therapeutic option for PROS patientsfor whom no medication currently exists.

Effect of BYL719 in Human Cells with PIK3CA Mutation

Next, we examined the efficacy of BYL719 at inhibiting the PIK3CApathway in human cells. To this aim, we used T-47D human cells derivedfrom breast cancer. These cells harbor heterozygous mutation of thePIK3CA c.3140A>G (H1047R). This gain of function mutation is also themost frequent mutation observed in PROS patients⁶.

We first confirmed in these cells, the spontaneous activation of theAKT/mTORC pathway as assessed by the phosphorylation status of AKT onthe residue Thr³⁰⁸ and Ser⁴⁷³ but also the phosphorylation of the S6RPprotein (data not shown). Then, cells were exposed to increasingconcentrations of BYL719. We observed that, after 2 hours of BYLexposure at a concentration of 1 μmol/L, the AKT/mTORC pathway was fullyinhibited (data not shown). The effect was similar when the cells wereexposed to 4 and 6 hours to BYL719.

These data suggested that BYL719 is able to inhibit the PI3KCAactivation in human cells with PIK3CA c.3140A>G (H1047R) mutation.

Dramatic Impact of BYL719 in Patients with PROS

Based on these results, we decided to administer BYL719 as acompassionate use for two patients, one adult and one child, whosuffered from extremely severe clinical manifestation of CLOVES syndromeand were experiencing therapeutic failure and life threateningcomplications. Patient 1 was a 29 years old man with mosaicism andbiopsy proven gain of function mutation PIK3CA c.3140A>G (H1047R). Hehad overgrowth syndrome characterized by left leg hypertrophy,scoliosis, multiple naevi and extremely severe vascular abnormalities(data not shown). He underwent multiple aggressive debulking surgeriesto remove abdominal and dorsal vascular tumors, as well as multipleangiographies with embolization to limit tumor growth. He becameparaplegic at the age of 20 years old due to spinal cord compression andrequired bladder stenting to drain his urine. He developed progressivelyworsening severe systolic heart failure with a measured cardiac outputof 18 l/min and a Brain Natriuretic Peptide (BNP) level constantly over2500 pg/mL (N<100 pg/mL), resistant to all usual drugs. It is unclearwhether the heart failure was due to the presence of vascular shunts,PIK3CA mutation in the cardiac cells, or both. During 5 years, thepatient received rapamycin to limit tumor growth progression but withoutany efficiency. Lastly, the patient developed kidney dysfunction withsevere proteinuria. Kidney biopsy revealed glomerular lesions withextensive fibrosis. Kidney lesions may be the result of heartdysfunction, the use of rapamycin¹¹, and/or PIK3CA mutation in kidneyepithelial cells. CT scan (data not shown) and MRI (data not shown)showed severe vascular abnormalities, while PET-scan was negative. Dueto the severity of the case with poor prognosis, physicians, surgeonsand radiologists decided to stop any interventional treatment and toprovide him only supportive and palliative care. His vital prognosis wasengaged with survival estimated at few months.

Following our experimental findings and after ethical consideration wedecided to purpose the BYL719 to our patient. We obtained theapprobation from Novartis and the French regulatory agency (Agencenationale de sécurité du medicament et des produits de sante, ANSM) todeliver the BYL719 as a compassionate use. We stopped rapamycin and onemonth later we started the BYL719. The drug was orally delivered everyday. We choose 250 mg per day as a starting dosage, the lowest dose usedin clinical trial and based on our in vitro data. Within the first fewdays we observed a dramatic improvement of the general status of thepatient. The patient started to feel better and more comfortable. Weekafter week we could objective a dramatic effect of the drug on tumorssizes, venous dilation and skin aspect (data not shown). He lost 23 kgin 12 months due to edema clearance but also due to a major reduction ofall vascular tumor abnormalities (data not shown). The circumference ofthe thorax and the abdomen reduced dramatically, A-20% and A-35% in 12months respectively (FIG. 2A). CT-scan and MRI confirmed global vasculartumors shrinkage with a 63% volume decreased at 12 months, as well assubcutaneous infiltration disappearance (data not shown). The effect ofthe drug on the heart function was remarkable, with a completecorrection of the BNP level in the course of 4 weeks (FIG. 2B). Thecardiac output dropped to 3 l/min, and the size of the heart reduced of25% as determined by CT scan (data not shown). Left ventricular massindexed to body surface area decreased from 250 g/m² to 148 g/m². Renalfunction was also rapidly improved with an estimated glomerularfiltration rate increasing from 33 to 52 ml/min/1.75 m² (FIG. 3D). Thevolume of the hypertrophic left leg also reduced (data not shown). Weobserved a dramatic improvement of the skin hypertrophy with a change inthe nevus coloration and a reduction of the size of the ear (data notshown). Lastly, 6 months after BYL719 initiation the patient began tohave partial gain bladder function with improvement of saddleanesthesia. MRI revealed a 60% reduction in size of the venousmalformation that was compressing the spinal cord. Twelve month later,the patient had no side effect except hyperglycemia that was wellcontrolled by regimen. The patient is still receiving the BYL719.

Patient 2 was a 9 years old girl with a CLOVES syndrome diagnosed twoyears prior to this study and biopsy verified gain of function mosaicismmutation in PIK3CA c.3140A>G (H1047R). She had scoliosis, left leghypertrophy, vascular abnormalities and hypertrophic muscle of the back(data not shown). Importantly, she had a voluminous cystic lymphangiomainvolving the left kidney and the gastrointestinal tract. PET-scanshowed metabolic fixation of the thymus, muscle from the back and theleft leg (data not shown). The intraabdominal tumor was growing rapidly,with surgical or interventional radiology deemed impossible. Afterethical consideration, we obtained the approbation from Novartis and theFrench regulatory agency (ANSM) to provide BYL719. Since the drug hasnever been tested in a child, we decided to start at the lowestavailable dosage, 50 mg/day. As with the first patient, we observed adramatic clinical improvement rapidly after the beginning treatment(data not shown). The patient reported improved comfort and we observeda 15% reduction in the volume of the left leg, the left foot (1 shoesize reduction), and 8% of her abdominal circumference (data not shown)in 120 days. Hypertrophic muscles of the back shrunk rapidly and,unexpectedly, her scoliosis was reversed in 120 days without any otherintervention (FIG. 3). More importantly, MRI showed a 60% reduction ofthe volume of the intraabdominal tumor in only 4 months. PET-scanperformed 4 months after treatment initiation confirmed the reduction ofthe disease in almost all tissues. During the follow up, we found thenormal growth of this patient was not impacted by the BYL719, and shereported no side effects. The patient is still receiving treatment.

DISCUSSION

Here, we report the first mouse model of PROS that recapitulates thehuman phenotype, a rescue of PROS in the mouse model by treatment withthe PI3CA inhibitor BYL719, and, using a translational approach, havedemonstrated the efficacy of BYL719 in PROS patients. This studyprovides the first direct evidence supporting PIK3CA inhibition as arobust and effective therapeutic strategy in the previously untreatablePROS.

BYL719 treatment promoted the recovery of two patients, an adult and achild, with positive clinical impact observable rapidly after initiationof treatment. As in the mouse model, the drug improved all organdysfunctions related to PROS. Importantly, the drug maintained efficacyin improving malformations during the entire treatment period. The drugwas well tolerated, without any significant side effects, in contrast tomTOR inhibitors. As anticipated, given that PI3KCA is downstream of theinsulin receptor, hyperglycemia occurred in the first patient,indicating that caution should be exercised given the potential forlong-term side effects, particularly in children where growth,fertility, and puberty could be affected. However, during the treatmentperiod the growth of the child was not affected, and any detrimentaleffects must be weighed against the benefits in this devastating andpreviously intractable disease. Several questions remain, including whenthe best period is to start the treatment and when or if it therapy canbe halted. Given the mouse data it seems highly possible that withdrawof the drug would be associated with the restart of the diseases, as isalso seen in patients with Tuberous Sclerosis¹⁷.

PIK3CA is one of the most commonly mutated oncogenes in human cancers,and similar mutations are found in PROS patients⁶. As in cancer,although PIK3CA mutations can be detected across the entire codingsequence of the gene, most of the mutations are found in 3 major hotspotclusters in the helical (E542K, E545K) and kinase domains (H1047R). Eachof these mutations leads to gain-of-function activation of PIK3CApathway. Importantly, in cancer models, BYL719 is able to fully blockthe activity of the pathway regardless of the type of mutation¹⁴.Additionally, our mouse model of PROS based on an increased number ofcopy of the PIK3CA gene, is rescued by the BYL719. Taking together thesedata suggest that the drug is certainly efficient in all form of PROSand not only acting on the H1047R mutation.

This discovery may have broad clinical impact. Importantly, if PROS arerare, they are not exceptional. The number of PROS patients is assumedto be largely underestimated due to phenotype heterogeneity leading tomisdiagnosis. Mosaicism often requires the use highly sensitivetechnologies to detect PIK3CA somatic alteration⁶. With an incidence of1/100,000 the recent proposal to include patients with Klippel-Trenaunaysyndrome into the PROS highlights the great number of patients that maybenefit from BYL719⁹. Importantly, PROS is an evolving entity since moreand more diseases, such as isolated venous malformations can also belinked to PIK3CA mutations^(8,18,19).

Our mouse model offers a unique opportunity to better understand thephysiopathology of the PROS. This model recapitulates PROS withvasculature abnormalities, muscle hypertrophy, kidney dysfunction, liversteatosis, spleen disorganization and scoliosis. Severity was related tothe high percentage of Cre recombination events induced by tamoxifentreatment. The mice perished due to severe bleeding, particularly fromthe mesenteric and liver vessels. Bleeding and leakage are among thesevere complication observed in patients with CLOVES syndrome⁶. We alsofound that overexpression of the PIK3CA gene in the liver lead tosteatosis as in the genetic overactivation of the AKT/mTORC²⁰.Remarkably, the mice quickly developed scoliosis after Crerecombination. This lesion is certainly due to the development ofhypertrophic paraspinal-truncal soft tissue masses, since the BYL719 wasable to quickly reverse the abnormalities. The improvement of scoliosiswas also visible in the two patients with reduction in muscle masses andhypermetabolic signal. BYL719 was able to either prevent the developmentof any tissue abnormalities or to rescue all defects while disease wasachieved.

In conclusion, the results of this work indicate that PIK3CA inhibitionmay be a safe and effective means to improve health and quality of lifein PROS patients. Obviously, these results will have to be confirmed inlarger cohort of patients but, they represent the most promisingavailable therapy for patient with PROS.

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

1. A method of treating PIK3CA-Related Overgrowth Spectrum (PROS) in asubject in need thereof comprising a step of administrating the subjectwith a therapeutically effective amount of BYL719.
 2. The method oftreating according to claim 1, wherein the PROS disorder is Congenital,Lipomatous, Overgrowth, Vascular Malformations, Epidermal Nevi andSpinal/Skeletal Anomalies and/or Scoliosis (CLOVES) syndrome.
 3. Themethod of treating according to claim 1, wherein the PROS disorder isKlippel-Trenaunay syndrome.